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1. Introduction
Skin melanoma is a devastating disease, frequently diagnosed in human and dogs, accounting for 0,8% - 2% of all skin tumors in latter species [1].
Melanocytic tumours are common neoplasms in dogs, accounting for 4 to 7% of neoplastic lesions in general, and up to 7% of all malignant tumours [2,3]. They generally arise on the oral cavity (Figure 1), lip, skin (Figure 2) and digit, amongst other locations (Figure 3) [4].
Figure 1.
Canine oral melanoma (courtesy of Dr. Abel Fernandes).
Figure 2.
Canine cutaneous melanoma.
Figure 3.
Canine ocular melanoma.
Biologic behaviour of these tumours is often related to its location. Over 85% of melanocytic lesions located on haired skin are described as of benign behaviour. The majority of oral and mucocutaneous junction melanomas, with the exception of the eyelid, and 50% of digital melanomas originated from the nail bed are reported as malignant [5,6].
Cutaneous canine melanocytic lesions are usually benign [7,8]. They are generally detected at a late stage, when excision is rarely curative and metastasis is often detectable in regional lymph nodes [7]. Malignant tumors are found most frequently on the head, ventral abdomen, and scrotum [7]; in the last one, they represent 3,1% of all cutaneous malignant melanomas and 4,7% of scrotal tumours [9]. Amelanotic lesions can occur as cutaneous neoplasms, but are more frequent in the oral cavity, and tend to be behaviorally malignant [10].
Metastasis are often found on regional lymph nodes and lungs, but organs as brain, heart and spleen are also commonly affected [11].
Veterinary nomenclature of canine melanocytic tumours has been subjected to many controversies and changes over time. Through this article, in accordance with the revised World Health Organization classification system, and in order to simplify and avoid confusion, the authors describe benign lesions as melanocytoma, whereas malignant lesions are referred to as melanoma [12].
Etiology of this neoplasms is still uncertain, and several factors may be related, such as consanguinity, trauma, chemical exposure, hormones and genetic susceptibility [10]. Unlike humans, ionizing solar radiation exposure doesn’t seem to be related to canine melanoma initiation [4].
Canine melanocytic tumour diagnosis often represents a challenge for the pathologist, since a high number of neoplastic lesions are quite similar in terms of its clinical and histologic appearance, including carcinoma, sarcoma, lymphoma and plasmocitoma, amongst others [10]. Indeed, cutaneous neoplasms with malignant behavior are more difficult to distinguish histologically from benign neoplasms than oral or lip neoplasms [13].
Diagnosis is usually based on fine-needle aspiration citology, but biopsy for histopathological examination is essential to determine its malignant potential [4,14]. The most reliable histologic criteria is the mitotic index, defined as the total number of mitotic figures observed per ten high-power light microscopic fields, which is known to be 90% accurate [5,6,15].
Immunochemistry has arised as an extremely useful tool, for both diagnostic and prognostic purposes. A positive diagnostic for melanocytic neoplasms is obtained with a positive labelling of S100 protein, vimentine, Neuron Specific Enolase (
The treatment of choice for local cutaneous melanomas is surgical excision; tumours with benign histopathology criteria have an excellent prognostic after this surgery. However, for malignant tumors, the prognostic is guarded, since metastatic rates of 30-75% have been reported [5,6].
Alternative therapy methods described in the literature include systemic chemotherapy, radiotherapy [17], photodynamic therapy [18], local hyperthermia [19,20] and intralesional injection of cisplatin or carboplatin.
The poor responses to the conventional therapy are leading to a development of new immunotherapy procedures – including intralesional adenoviral vector-mediated transfer of CD40L, a tumor necrosis factor gene [21], therapy with a plasmid DNA encoding staphylococcal enterotoxin B [22], and systemic tratment with liposome-encapsulated muramyl tripeptide [23].
At last, the most promising therapy appears to be a xenogenetic human tyrosinase DNA vaccine, with minimal local reaction and no systemic toxicity signs, and a great clinical responde with significant increasing of the survival time [24].
In this paper, the authors aim to contribute to the understanding of melanocytic tumours in the dog, making a critical review of the literature and discussing the parameters currently considered valid for diagnosis use in canine melanocytic neoplams.
2. Signalment
Cutaneous melanocytic tumours are most common in older dogs (mean age of 9 years old) [25], with a higher mean age for dogs with malignant melanocytic tumours (12 years). However, age has not been related with the patient clinical outcome and survival time [26].
Although all breeds of dog (and crossbred animals) may be affected, some breeds are reported as predisposed, including Schnauzer, Doberman, Scottish Terrier, Irish Setter, Golden Retriever, Chow Chow, Cocker Spaniel, German Shepherd and Rottweiler [4,27]. Breed predisposition is thought to be related to an underlying genetic risk and/or increased pigmentation in the described above breeds [4].
One study [6] has also established a relationship between patient breed and tumour behavior, likely due to genetic susceptibility, as prior described. In the referred work, melanocytic lesions tended to be behaviorally benign in Doberman pinschers and miniature schnauzers, while miniature poodles were the mostly affected breed with malignant melanoma. However, it must be noted that oral neoplasms were also included in that study.
An early report described a higher frequency of these lesions in male dogs [28], but recent literature denies gender predisposition [6,27,29-31].
3. Pathogenesis
Melanin is a dark-brown pigment synthetized by melanocytes, dendritic cells found within the basal layer of the epidermis. These cells are dispersed from each other, located between basal keratinocytes, forming adherent and regulatory junctions mediated by epithelial cadherin (E-cadherin) molecules. After its synthesis, melanin is retained in melanosomas and transferred to the adjacent keratinocytes [32].
Conversion of normal melanocytes to clusters of neoplastic melanocytes is a process composed by a series of events: initiation, promotion, transformation and metastasis [4].
Little is known about initiation on most animal melanomas, but ionizing solar radiation exposure – the main initiator factor in Human melanomas [33] - doesn’t seem to be related to canine melanomas [4]. A higher incidence of spontaneously mutated cells due to familiar clustering through inbreeding may be a critical initiation factor in domestic animals.
Malignant transformation of canine cutaneous melanocytomas is very uncommon. Regarding cutaneous melanomas, there are a few published case reports, including one by Valentine and team [34], which have described a single case of malignant transformation of a congenital melanocytic nevus is a Golden retriever. Conroy [35] described two cases of melanoma originated from junctional or dermal hamartomas and a single case of a primary melanomas originated from a subcutaneous melanocytoma have also been reported [36]. In summary, canine cutaneous melanomas are thought to arise
Promotion phase is related to mutated cells proliferation, with subsequent amplification of cell population and origin of additional mutations [4]. Melanoma promoters include chronic trauma, chemical exposure, drugs and hormones [10], and its action results in reactive hyperplasia of the epithelium, with disruption of regular keratinocyte-melanocyte interactions and proliferation of initiated cells.
The next step in carcinogenesis involves a series of transformation events. Recent developments in genetic and molecular study techniques have identified the role of a few tumour suppressors in melanoma cell lines, giving new insights on the importance of these molecules in canine melanoma development. A reduction or loss of
After local proliferation phase, neoplastic cells may acquire a malignant behavior, and disseminate through hematic or lymphatic vessels to various other organs, originating secondary neoplasms known as metastasis. This complex process has it start with loss of adhesion and detachment of neoplastic cells from the primary mass, hematic and lymphatic vessels intravasion and attachment and proliferation within a secondary location [4].
Metastasis process is dependent of various adhesion molecules regulation by neoplastic cells. Several studies have shown an association between decreased and altered expression of E-cadherin, a calcium-dependent adhesion molecule responsible for melanocyte-keratinocyte interaction, and canine cutaneous melanoma progression [39,40]. CD44, a second transmembrane glycoprotein which facilitates metastasis, is required for several processes, including hyaluronate degradation, cell aggregation and migration, angiogenesis and hematopoiesis [4]. Down-regulation of regular CD44 plus up-regulation of CD44v5 has also been associated with melanoma metastasis, particularly with lymph node metastasis [41].
Autonomous growth is a key requirement for both primary and secondary neoplastic development. The most important autocrine growth factors in animal melanoma include basic fibroblast growth factor (
4. Gross morphologic features
Macroscopically, canine malignant melanoma cannot be differentiated from melanocytoma [42]. Melanomas in dogs tend to be dermal in location, unlike Human melanomas – which are intraepidermal with some degree of dermal invasion. Prognostic schemes, such as Clark’s level or Breslow thickness, built nased on depht of dermal invasion, are not applicable on canine lesions [43].
Canine melanocytomas share some aspects with Human benign melanocytic lesions, in terms of clinical evolution most common metastatic locations [42], and genetic alterations [44].
Cutaneous melanocytomas are usually symmetrical, circumscribed, but encapsulated [43], solitary, black, brown, or gray cutaneous alopecic nodules [43,45] with a variable size with range of 1-4 cm in diameter (Figure 4 and Figure 5) [43]. Epidermis is usually intact, and alopecia is frequent. Epidermal cells may be hyperpigmented, and the majority of dermal cells are replaced by the tumoral ones, which in larger masses might also extends into the subcutaneous tissue. The tumors may have a varieated appearance, with areas of pigmentation intermingled with no pigmented regions [42].
Canine cutaneous malignant melanomas can vary considerably in appearance, regardless of the location. Melanomas tend to be asymmetrical. The asymmetry may be most readily recognizable in the epidermal component of junctional tumors [43]. Melanomas size vary from some milimiteres to as large as 10 centimeters in diameter (mean range being 1 to 3 cm in diameter) [43], but this is not a reliable indicator of malignancy [13,42]. The color is variable, ranging from gray or brown to black, red, or even dark blue [7]. Cutaneous melanoma presentation includes smooth domes, sessile nodules, polypoid, plaquelike [7,43], or even lobulated masses [7]. The larger ones are often ulcerated [7,43]. The tumors may invade deeply into the subcutaneous tissue and along fascial planes [42].
Figure 4.
Canine cutaneous melanoma.
Figure 5.
Canine cutaneous melanoma.
5. Cytological diagnosis
Microscopic examination of a cytological specimen obtained through fine needle aspiration has become a valuable technique to obtain a preliminary, and often definitive, diagnosis [46]. Being a quick, non-evasive and inexpensive procedure, it can also provide information on the stage, prognosis and metastasis evidence. Its main limitation reside on the fact that non-pigmented melanomas may strongly resemble other neoplastic lesions, and the amount of cytological sample might be very small and not fully representative of the lesion [4,46].
Several studies in Human cancer have described a strong accuracy in cytological examination in comparison with histopathological findings [47,48], but there are only few studies on the subject in Veterinary Medicine [49]. For instance, Ghisleni and others [50] evaluated a series of cutaneous and subcutaneous masses from dogs and cats through histopathology and cytology, describing an agreement between both techniques in 90.9% of the samples.
Melanocytic tumours are characterized by the presence of cells with abundant cytoplasmatic melanin granules. Neoplastic cells may appear with an epithelial (cohesive cells), mesenchymal (single oval or spindle-shaped cells) or round cell morphology. Nuclei may present a central or eccentric location, and is often solitary, though multinucleated forms are occasionally found. Nucleoli tends to be very prominent, with variable shapes such as round, oval or angular. These cells have a light basophilic cytoplasm, with a moderate to high nuclear-cytoplasmatic ratio. Varying degrees of pigmentation might be found within the same tumour smear [4,46], (Figure 6 and Figure 7).
Malignant criteria consist, most importantly, of marked anisokaryosis and nuclear pleomorphism, but also of the presence of large and atypical nucleoli [46]. Mitotic index, the most reliable criteria in histopathological evaluation, has no use in cytology. Regional lymph nodes are the most commonly evaluated site while monitoring for metastasis [4].
Figure 6.
Canine cutaneous melanoma (Wright, 100x).
Figure 7.
Canine cutaneous melanoma (Wright, 400x).
6. Histological diagnosis
Histological characteristics of canine melanocytic neoplasms were defined by World Health Organization, in
Figure 8.
Canine cutaneous melanocytoma (H&E, 200x).
Figure 9.
Canine cutaneous melanoma (H&E, 200x).
Histological appearance does not always correlate well with biological behavior [8]. More recent studies have provided a numerical ‘‘tumor score’’ taking into consideration mitotic index, nuclear atypia, inflammation, necrosis and volume which appears to have improved correlation between histology and behavior [8].
The term nevus, commonly used in describing pigmented melanocytic lesions of the epidermis and dermis in humans, is not used in veterinary dermatopathology [7].
In this chapter we review several histological parameters on their ability to diagnose and predict prognosis (i.e., prediction of mortality) of canine melanocytic neoplasms. Malignancy celular features include a characteristic large nucleous, nuclear atypia, hyperchromasia, abnormal chromatin clumping and anomalous mitotic figures [51].
6.1. Predominant cell type
Melanocytic neoplasms are generally composed of one of the following cell types: epithelioid (Figure 10), spindle, mixed (Figure 11), dendritic [51,52], and round cells [43]. Other less commonly described cell types include signet ring and ballon cells [51]. All these cell types may occur either alone or in combination [43]. In melanomas, ganglion cell and multinucleated giant cell forms also may be observed [43]. The epithelioid cell type is the most common type in all locations, whereas a mixture of cell types is seen with less frequency [51].
The epithelioid cells are round, with discrete cell borders, abundant glassy cytoplasm, appearing arranged in sheets and larger nests [43]. Similar to their spindle-shaped counterparts melanomas may exhibit large ovoid nuclei and prominent nucleoli [7,43], marked anisokaryosis and variable chromatin patterns [43].
The spindle cell tumors are arranged in streams and interweaving bundles, resembling fibrosarcoma or neurofibrosarcoma presentation. In malignant lesions, the nuclei are large and fusiform with prominent nucleoli [7,43], and moderate to marked nuclear pleomorphism is seen [43]. Spindle cell predominant morphology was statistically associated with benignity in one study (in 71% melanocytomas in contrast to 29% melanomas) [43]. The mixed type consists of both cell morphologies and patterns [7].
Dendritic melanocytes have a highly angular shape, sometimes with long cytoplasmic processes, and are usually arranged in small nests [43] or organized in tightly swirling streams, often with a fingerprint pattern [7]. The dendritic or whorled forms occurs only in the skin [53].
Round cells tumors have round to polygonal cells arranged in sheets as dense packets of cells, the packets being separated by a scant stroma. The nuclei is large and round in melanomas [43].
The signet-ring cell tumors consist of compact neoplastic clusters, with round to ovoid cells presenting a faintly-eosinophilic cytoplasm and an intensely-stained periphery. A vesicular nuclei, crescent-shaped, and with a peripheral location, gives the cell a signet-ring appearance [52].
Figure 10.
Epithelioid cell canine cutaneous melanoma (H&E, 400x).
Figure 11.
Mixed cell canine cutaneous melanoma (H&E, 400x).
Balloon cells are found organized in groups, separated by collagenous septa [52]. The cells are round to polyhedral and have clear or faintly eosinophilic cytoplasm [43,52]. In melanomas, the nuclei are round situated mainly at the periphery of the tumor cells [52]. Usually contains one central prominent nucleolus [7], which sometimes is difficult to detect. Heterochromatin, which is sparse, is dispersed throughout the nuclei [52].
Benign melanocytic cells present an enlarged vesicular nuclei with small nucleoli. Nuclear shape vary on according to the predominant tumour cell type. Mitotic figures are rare, and mitotic atypia is rarely observed. [43].
Although cell type appears to play some role in the prognosis of ocular melanocytic neoplasms, this feature lacked significance in prognosis of melanocytic tumors occurring in the mouth, feet, buccal mucosa and skin of dogs in several studies [8]. On the contrary, the epithelioid shape was associated with an unfavorable course, for 8/15 cases. The difference seems to be significant (p = 0,03) [12].
6.2. Nuclear atypia
One of the major criteria of malignancy in melanocytic neoplasms arising at any pigmented anatomic site is nuclear atypia. This feature is more valuable in epithelioid tumours than in spindle, whorled type or signet-ring cells, due to the insufficient nuclear detail associated with the later neoplasms [8,44,54]. However, not all studies are consensual [12].
Well-differentiated tumoral melanocytes have a small nucleus with one central nucleolus [8]. In contrary, undifferentiated tumours generally present cells with multiple, large, irregular and eccentrically nucleoli [8].
Several criteria are used to estimate nuclear atypia, including the percentage of nuclei involved [8]. Figures 12 and 13 (below) present moderate and severe nuclear atypia, respectively.
6.3. Mitotic index
In animal cutaneous and eye melanocytic neoplasms, mitotic index (MI) is the most reliable histological feature for distinguishing malignant from benign tumors [44,51], and also in predicting the clinical course of the disease [8]. In cutaneous melanoma, an MI of ≥3/10 hpf is significantly correlated with decreased survival [55].
The number of mitoses is usually lower in melanocytomas [<3 mitotic figures per 10 high power fields (hpf)] than melanomas [42,44,53].
In one study, the mitotic index was strongly correlated with the clinical outcome of tumors. For tumors with a favorable outcome, the mean value of the number of mitosis (on 10 randomly selected high power fields) was 1,98 (from 0 to 27). For tumors with a malignant behavior, it was 18,53 (from 0 to 75) [12].
The evaluation of the MI in conjunction with nuclear atypia classification (Figure 13) offers a more precise value the histological diagnosis [8].
Figure 12.
Canine cutaneous melanoma with moderate diferenciation (H&E, 400x).
Figure 13.
Canine cutaneous melanoma with evident nuclear atypia and numerous mitosis (arrow), (H&E, 400x).
6.4. Cellular pleomorphism
Cellular pleomorphism criteria include several features, such as cell size and shape, pigmentation degree and nuclear features (including prominence of nucleoli and chromatin pattern). The usefulness of these parameters as individual prognostic factors is doubtful; however, it increases when these are used together [8,27].
6.5. Degree of pigmentation
Degree of pigmentation is highly variable, even within a single smear (Figures 14, 15 and 16). Even on histological evaluation of amelanotic-classified tumours is common to detect a few very fine pigment granules in some cells. These are generally punctuate, spherical or elongated, as observed in pigmented keratinocytes. Cells with a very fine pigment may present a dusty gray appearance, instead of the typical granulation image [7]. In summary, it can be difficult to accurately diagnose an amelanotic melanocytic neoplasm and to define the degree of pigmentation [13].
Individual tumoral cells possess a different amount of melanin, which granules are generally small and uniform in size within the same cell. Splindle, round to polygonal, and balloon cells have sparsely-distributed melanin granules, while large epithelioid and dendritic cells are known to have a higher degree of pigmentation [43].
The majority of canine melanocytomas (except for ballon cell ones) have marked to moderate melanin pigmentation overall, especially in the superficial aspect of the tumors [43]. Similarly to cellular morphology, the degree of pigmentation of neoplastic cells was not an indicator of prognosis [12].
Figure 14.
Canine cutaneous melanoma with marked pigmentation (H&E, 400x).
Figure 15.
Canine cutaneous melanoma with moderate pigmentation (H&E, 400x).
Figure 16.
Amelanotic melanoma (H&E, 400x).
6.6. Junctional activity
Junctional activity refers to the proliferation of neoplastic melanocytes at the interface between the epidermis and dermis or epithelium and submucosa [53].
The presence or absence of junctional activity is not specific to melanoma and often occurs in melanocytomas [7], however, malignant melanomas arising in the skin often show marked junctional activity, (Figure 17 and Figure 18) [42].
Junctional activity was not statistically associated with survival for skin neoplasms in one study [8]. In contrast, another work considered junctional activity as an independent prognostic factor (p =0,0239) for cutaneous melanocytic neoplasms, and found that its occurrence was associated with a longer survival time (p = 0,0046) [55].
Figure 17.
Canine cutaneous melanoma without junctional activity (H&E, 200x).
Figure 18.
Canine cutaneous melanoma with junctional activity (H&E, 400x).
6.7. Intraepithelial neoplastic cells
The presence of intraepidermal tumoural cells can be graded as absent, slight (25% of neoplastic melanocytes in epidermis), moderate or prominent (more than 50% of tumour cells are present in the epidermis) [44]. However, this feature appears not to be of prognostic significance [8].
In a recent study was found that the samples of canine melanomas presented medium to prominent scatter of intraepidermal melanocytes, lower pigmentation, and higher nesting of intraepidermal melanocytes, in comparison with melanocytomas [44].
In most melanocytomas, the overlying epidermis is hyperpigmented, regardless of the presence or absence of intraepidermal clusters of neoplastic melanocytes [43].
6.8. Ulceration
Canine melanomas are most frequently ulcerated [44] and particularly larger masses [43] than melanocytomas.
According to Laprie and team [55], ulceration might be taken as a prognostic marker for this neoplasms. In the referred work, the presence of an ulcerated epidermis was associated with a shorter survival time (p = 0,0023) and shown to be an independent prognostic factor (p = 0,0065) [55]. However, two other studies found no correlation between ulceration and clinical evolution of lip, nail bed [27] or cutaneous melanocytic neoplasms [12,27].
6.9. Level of infiltration/invasion
Melanocytic tumours strictly limited to the dermis, with a shallow depth, are associated with a greater survival time (p < 0,0001), and deep level of infiltration has been shown to be a significant prognostic factor (p = 0,0012) [55]. One other study that evaluated the level of invasion (in cutaneous and subcutaneous tissues) concluded that tumors confined to the superficial dermis were associated with a benign course in 94% of cases. On the other hand, tumors reaching the deep dermis and the subcutis showed a malignant behavior; however, the sample number were too low to allow for a conclusion [12].
6.10. Necrosis
Necrosis is a common feature, particularly in larger masses [43]. The presence of necrosis was correlated with malignancy and with a short survival time in a study set of 389 melanocytic neoplasms containing both benign and malignant lesions from various locations (mouth, feet and lip, skin) [8]. In other report with a set of 38 malignant melanomas from various locations, no correlation was found with survival time [54]. In summary, necrosis is considered of limited prognostic value in animals [7].
6.11. Morphologic classification
Melanocytomas include dermal, compound, and balloon cell tumors, as well as multiple dysplastic melanocytoma syndrome in dogs [43].
Dermal melanocytoma are strictly intradermal in location and larger tumours may extend into the subcutis [43]. Melanocytomas are generally composed by spindle cells disposed in bundles, nests and whorles, with a moderate cellular concentration and a lack of stromal collagen [43,45]. Melanophages might be dispersed throughout the tumoral nodule or in aggregates [43]. Mitotic figures are ocasionally seen (inferior to 1 per 10 high power fields), and mitotic atypia is not observed [43].
Some dermal melanocytomas are composed of epithelioid or dendritic cells that are heavily pigmented [43]. Nuclear morphology may be obscured by the large amount of pigment [43]. Although nuclei may be large, there is minimal nuclear pleomorphism [43].
A compound melanocytoma has a wedge-shaped configuration, and its description refers to the fact of including both junctional and dermal components. A numerous and densely packed tumor cell population is present in the dermis, while a variable amount of tumor cells accumulate in clusters and nests within the epidermis, along the dermal–epidermal junction and in the outer follicular wall – this pattern is referred to as ‘junctional activity’ [43].g
Balloon cell melanocytoma have a dermal location, and are predominantly composed of large round cells [43,45], although some fusiform or polygonal melanocytes might be present [42], with an abundant, pale eosinophilic and finely granular cytoplasm [42,43,45]. These lesions often lack readily visible pigmentation; however, dust-like melanin granules may be detected in small numbers [42,43]. Nuclei are small, uniform, and ovoid [43,45] and mitotic figures are rarely observed [42,43].
Multiple dysplastic melanocytoma syndrome mostly resemble compound melanocytomas on low magnification. However, their incidence increases in larger lesions. Also, cytologic atypia and mitotic figures are present and some of the proliferating melanocytes have irregularly shaped and enlarged hyperchromatic nuclei [43].
Canine melanocytoma–acanthomas are mixed tumors that are composed of a benign melanocytic proliferation, resembling compound melanocytoma, and a benign epithelial proliferation [43,45]. The epithelial component usually appears follicular and resembles an isthmus-type tricholemmoma or infundibular keratinizing acanthoma [43]. The epithelial population forms a mass in the dermis composed of cords and nests with occasional small cystic structures containing keratin [45]. Melanocytic cells form nests in the epidermis and sometimes in the cords of epithelial cells within the dermal mass; melanocytic spindle cells can form whorls and bundles between the epithelial cords and nests [45]. A dermal melanocytoma– acanthoma has been immunophenotyped in a German Shepherd dog identifying the presence of keratinocytes and melanocytes [56].
Dermal melanomas have no junctional activity, but in some cases the tumour might extend deeply into the subcutaneous tissue. There might be a predominance of a certain cell type, but most tumors reveal a mixture of spindle cells, round to polygonal cells, and/or epithelioid cells. While spindle cells are poorly pigmented, more round and epithelioid cells tend to have a moderate to abundant amont of melanin granule [43]. Other important features in this kind of neoplastic lesions include a marked nuclear pleomorphism, nucleolar prominence, a moderate mitotic rate (3 or greater per 10 high power fields), atypical mitotic figures, asymmetry of the tumor nodule and a lymphoplasmacytic cell population [43].
Melanomas have an obvious ‘junctional activity’ pattern, with tumor cells distributed through the dermal–epidermal junction, as well as at higher levels of the epidermis, particularly in those of the nail bed and lip [43]. The intraepidermal element is mainly composed of epithelioid melanocytes, disposed individually or arranged in nests and clusters [43]. Tumours with numerous melanocytes distributed through all levels of the epidermis are referred to as lesions with a ‘pagetoid’ pattern [43]. Other melanomas present numerous individual neoplastic melanocytes present within the basal cell layer only, which is also referred to as an ‘atypical lentiginous infiltrate’ [43].
Spindle cell and desmoplastic melanomas, a subgroup of dermal melanomas, is composed predominantly of spindled melanocytes densely packed, or arranged loosely within abundant pale stroma [43]. Occasionally, there is a prominent fibroblastic component associated with the spindle shaped melanocytes, and collagen may become more abundant than the tumor cells [43]. The vast majority are amelanotic, thus a Fontana–Masson stain usually is necessary to detect the presence of melanin granules; cells are usually arranged in bundles or palisades, mimicking tumors of neural origin [43].
Balloon cell melanoma (clear cell melanoma), possess large cells with a clear eosinophilic cytoplasm [43]. The majority of balloon cell melanomas are amelanotic [43]. Some dust-like melanin granules may be present in a few tumor cells, and Fontana–Masson staining may be required for their demonstration [43,45]. These cells have a large vesicular nuclei with a prominent nucleoli [7,43]. Mitotic activity is generally low [43,45] and these dermal masses exhibit no junctional activity [45].
Signet-ring melanomas are composed of round to polygonal cells [45,52], with a pale eosinophilic cytoplasm and a darker periphery [52]. The nuclei are vesicular, crescent shaped and located at the periphery, giving the cells the appearance of signet-rings [52]. Nucleoli are prominent and occasional multinucleated cells may be present [45].
7. Histochemical diagnosis
The histological diagnosis of melanoma can be a challenge for the pathologist, especially in amelanotic tumours. On the other hand, in tumours heavily pigmented the observation of cellular features could be very difficult, requiring the use of bleaching, a histochemical method where melanin is extracted [7].
In cases where the diagnosis of a melanocytic tumor is not evident, histochemical methods specific for the cells producing melanin, such as DOPA (dihydroxyphenylalanine) reaction can be used [7,43].
8. Molecular diagnostic methods
A diagnosis of melanocytic tumors in dogs is not always easy to obtain only by conventional histological methods. Melanoma is often similar to other tumours types and has a highly variable histologic pattern which implies an accurate differential diagnosis. Furthermore, the distinction between benign and malignant tumors is not always easy [7]. Thus, it is essential the research of additional tools that can be used in melanocytic tumours diagnosis and to achieve a more accurate prognosis [57].
8.1. Classical diagnostic markers
Several markers are used to evaluate the presence of proteins normally found in melanocytes or in cells of neuroectoderm origin. The most common antibodies used are S-100 protein,
In the absence of an ideal marker that excludes definitively other tumour types, confirms a diagnosis of canine melanoma and positively reacts with tumour cells in all melanomas, a diagnostic panel must be performed, including different antibodies [58-61].
8.2. Growth fraction
The proliferative activity has provided valuable information on cell growth kinetics and consequently tumour behavior in melanocytic tumours [63]. There are some markers that estimate tumor proliferation, by identifying steps associated with cell cycle [64].
MIB-1 is the monoclonal antibody which is reactive against Ki-67 nuclear antigen, a protein present in all active phases of the cell cycle (G1, S, G2 and M) while being absent in resting phase (G0) [65,66].
In canine melanocytic tumours, Ki-67 may be useful in distinction between benign and malignant tumors: melanocytomas seem to have a significant lower growth fraction than malignant melanomas [67]. Furthermore, KI-67 could be an important prognosis factor in canine cutaneous melanocytic tumours [64,68].
8.3. DNA ploidy
Changes in DNA content may reflect chromosomal alterations and represent tumour genetic instability [69-72]. Changes in the DNA ploidy constitute an early event in carcinogenesis [73,74] and detection of aneuploid cell population of pre-neoplastic lesions can be considered a factor risk of the emergence of malignant tumours [75]. Although usually benign tumors were diploid [76] and aneuploidy were malignant [77], diploidy is not synonymous of a benign behavior [78]. Moreover, not all malignant tumors are aneuploid [79,80].
There are few studies about the ploidy assessment in canine melanocytic tumors [81,82] and its usefulness is still discussed. DNA index and ploidy balance seem to provide an additional tool to evaluate melanocytic tumors, being useful in the distinction of benign and malignant melanocytic tumours, mainly in amelanotic lesions [82]. Flow cytometry apparently has a limited utility for predicting the biological behavior of pigmented canine melanomas. DNA content and nuclear morphometric variables have little value in predicting survival time [83].
8.4. c-kit expression
The c-Kit protein (CD117), a transmembrane receptor that belongs to RTK III family, is a growth factor for melanocyte migration and proliferation. A loss-of-function KIT mutation are usually related with human melanocytic tumors [84,85].
In canine cutaneous melanocytic tumours, c-kit immunolabeling (both extension and intensity) were generally higher in melanocytomas than in malignant melanomas. The lack of c-kit expression in canine cutaneous malignant melanomas might be used as a criteria of tumor aggressiveness, helping to achieve a proper diagnosis [86].
8.5. Matrix metalloproteinase 2 and 9 expression
MMPs are zinc- and calcium-dependent proteases that promove not only the disruption and remodeling of structural barriers [87-89] but also a response to signaling molecules, acting as ligand for cellular adhesion receptors [90-92].
MMP-2 is widely distributed and constitutively expressed by most cells [93,94]. This protease has major roles reducing cell adhesion, stimulating cell migration and differentiation, and acting as an anti-inflammatory factor [95]. MMP-9 expression is normally induced, while almost MMPs are constitutively secreted after their translation [93,96], and may act as anti- or pro-inflammatory factors [95].
MMPs play a pivotal role in cancer development and progression [92,94,96-98] contributing to tumour proliferation, invasion, intravasation into circulation, extravasation, migration to metastatic sites and angiogenesis [90,94,99-101], deregulate the balance between growth and antigrowth signals in the tumour microenvironment [97,102,103], orchestrate inflammation [94,102,104], and evade apoptosis [89,91,102].
In canine cutaneous melanocytic tumours, MMP-2 and MMP-9 may be taken as a complement to histology in tumour diagnosis, especially in borderline lesions. Both MMP-2 and MMP-9 were expressed in the majority of canine cutaneous melanocytic tumours. MMP-2 is most commonly expressed in melanocytomas than in melanomas [105]. MMP-9 was overexpressed in malignant melanomas, compared with its expression in melanocytomas [106].
Additionally, in canine malignant melanomas a switch may occur in the MMP expression profile during tumour progression; meaning that the aggressiveness, evaluated by nuclear grade, seems to be associated with a decrease of MMP-9 and an increase of MMP-2 expression [105].
8.6. Inflammatory cells associated to tumour
The tumour associated inflammatory infiltrate may be modulate and determine tumor behavior [107]. The most studied cells in CCMT are macrophages and T-lymphocyte, however, studies on the matter are scarce.
8.6.1. Tumour Associated Macrophages (TAMs)
Macrophages constitute the most abundant leukocytes in the tumor environment, recruited by a number of chemoattractants that are produced by the tumor cells and tumor-associated stroma [108,109]. TAMs play a critical role in tumour progression and invasion by inducing neovascularization, suppressing immunocompetent cells and supporting cancer stem cells [110-113].
One study published by our group found that canine cutaneous melanocytomas present a lower number of TAMs than malignant melanomas. TAMs could constitute an important marker of canine melanocytic aggressiveness, being implicated in the progression of melanocytic precursor lesions to malignant melanoma [114].
8.6.2. T-Lymphocytic Infiltrate (TLI)
In spite of the fact that the role of the T-lymphocytic infiltrate in cancer tumourigenesis remains controversial, several studies showed that the presence of TILs are related to tumoural behavior in different tumour types [115,116].
Preliminary studies of our group showed that there is a difference between TILs in benign and malignant melanocytic neoplasms, whereas all melanocytomas presented little or even absence of TILs and melanomas had a more intense TILs, (Figure 19), [117].
Figure 19.
Abundant CD3+TILs in canine cutaneous melanoma (IHC, 100x), courtesy of Dr. Patricia Monteiro.
8.7. E-cadherin/β-catenin
Epithelial cadherin (E-cadherin) is a transmembranar glycoprotein which belongs to a family of cell to cell adhesion molecules dependent of the present of calcium molecules to bind cytoskeleton proteins through catenins. A decreased or altered expression of E-cadherin molecules often represents an increased invasiveness of tumour cells and ultimately malignancy in animal and human epithelial tumours [118,119].
In canine melanocytic tumours, the benign lesions present a membranous labelling (Figure 20) and the malignant ones an erroneous labelling, with a cytoplasmatic predominant immunostaining. Additionally, a loss of E-cadherin expression is noted in melanoma [120]. A loss of membrane E-cadherin/β-catenin complex is also detected in canine melanoma showing that a disruption of E-cadherin/β-catenin complexes and a increase of β-catenin may be associated with canine melanocytic tumours progression and aggressiveness [121].
Figure 20.
Strong membranar E-cadherin expression (IHC, 200x), courtesy of Dr. Mariana Santos.
8.8. Cox-1 and Cox-2 expression
Cyclooxygenase (COX), also known as the prostaglandin H-synthase, is an enzyme envolved on prostanoids biosynthesis. Cox-1 and Cox-2 are the two cyclooxigenase isoforms identified to date, similar in its structure but produced by different genes. The biological functions are also different: Cox-1, constitutively expressed in many tissues, plays an important role in the regulation of normal physiological, while Cox-2 is usually absent from normal cells but induced by growth factors, inflammatory reactions, tumour promoters and oncogenes [122-125].
Cox-1 and Cox-2 expression was recently described in canine melanocytic tumours [126-128]. Cox-1 is expressed in almost every tumours, both benign and malignant melanocytic skin lesions. Regarding Cox-2, melanocytomas did not present a positive immunolabelling, but in melanomas Cox-2 expression was present in more than 50% of the tumours [114,127]. The differences observed suggest that Cox-2 expression could be a useful tool in canine melanoma diagnosis, particularly in borderline lesions.
COX-2 expression was also observed in tumours with epithelium ulceration, necrosis, high mitotic index and nuclear grade and in less pigmented neoplasms, which could represent the higher aggressiveness of Cox-2 positive melanocytic tumours. In canine malignant melanomas, Cox-2 is associated with a higher cellular proliferation [114]. Besides the relation with tumour behavior [127], Cox-2 over-expression relates with a poor overall survival [129].
8.9. Angiogenesis
Vascular system is essential in oxygen and nutrients supply, elimination of metabolism products and promoting efficient access of leukocytes [130].
Angiogenesis is a complex process by which new blood vessels develop from pre-existing vasculature [131-133]. It is a fundamental requirement for organs development. Angiogenesis is also implicated in the pathogenesis of different pathological alterations, such as cancer and inflammation [134-136].
The tumor-associated neovascularization, by establishing continuity with the systemic circulation, allows tumor cells expressing their critical advantage in growth and facilitates metastization [137-139].
8.9.1. Vascular endothelial growth factor
Angiogenesis is a delicate process, tightly regulated by the balance of pro- and anti-angiogenic factors [140]. Among the angiogenic factors, VEGF is the more powerful and ubiquitous vascular endothelial growth factor, capable of inducing proliferation, migration, specialization and survival of endothelial cells [141,142]. Functions of VEGF family members related to neoplastic pathogenesis are linked not only to its angiogenic capacity [143], but also with the lymphangiogenesis [144,145], immunosuppression [146-148], stimulation and recruitment of endothelial and hematopoietic precursors of the bone marrow [149,150] and anti-apoptotic activity [150].
In canine melanomas, the only study published on VEGF expression is in oral melanomas. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy and were associated with tumour stage [151].
This is a promising area, since VEGF may be a good indicator of preneoplastic change in melanocytic lesions [137,152]. VEGF plays a role in human melanoma progression [153,154] with a strong involvment in the switch from the radial to the vertical growth phase and the metastatic phase. So, anti-angiogenic agents might even interfere with or block melanoma progression [155].
Preliminary studies performed by Gomes J and Pires I (data unpublished) show that VEGF may be useful as a discriminating factor between malignant melanoma and benign, since it is more intensely expressed in melanomas (Figure 21) than in melanocytomas.
Figure 21.
Canine cutaneous melanoma with a strong and diffuse VEGF expression (IHC, 200x), courtesy of Dr. Joana Gomes.
8.9.2. Microvessel Density (MVD)
Tumor angiogenesis can be estimated through a quantification of microvessel density (MVD). The most widely used method is the immunohistochemical methods, in which specific markers for endothelial cells are employed, as von Willebrand factor (Figure 22), CD34 and CD31 [156-158].
MVD seems to be important for diagnostic purposes in canine MT’s. MVD is significantly higher in melanoma than in melanocytomas [159] and its expression has been associated with a high mitotic index, necrosis and ulceration (study performed by Gomes J and Pires I, data unpublished). However, its prognostic significance is still discussable [159,160].
Figure 22.
Tumoural neovessels positive to von Willebrand factor (IHC, 400x), courtesy of Dr. Joana Gomes.
9. Conclusions
The incidence of melanoma is increasing annually both in man and in dog. Given that dogs and humans share the same environment and similarities between human and canine melanoma it is urgent to discuss common mechanisms in melanoma development in both species.
Melanoma diagnosis in dogs can be challenging due to the variety of its histological appearances, especially when pathologists are facing amelanotic or metastatic lesions. Although the definitive diagnosis of a melanoma is often difficult by the lack of specific markers that can distinguish these lesions, immunohistochemistry plays a key role in the differential diagnosis with other neoplasms.
Additionally, the distinction between benign and malignant melanocytic tumours is not always easy, especially in borderline lesions, thus the importance of a strong knowledge of new markers of malignancy for the establishment of a definitive diagnosis and a correct therapy managment and prognosis establishment.
References
- 1.
Grandi F, Rocha RM, Miot HA, Cogliati B, Rocha NS. Immunoexpression of S100A4 in canine skin melanomas and correlation with histopathological parameters. Veterinary Quarterly 2014 1-7. - 2.
Goldschmidt MH. Benign and malignant melanocytic neoplasms of domestic animals. The American Journal of Dermatopathology 1985;7 203-12. - 3.
Cotchin E. Melanotic tumours of dogs. Journal of Comparative Pathology and Therapeutics 1955;65 115-IN14. - 4.
Smith SH, Goldschmidt MH, McManus PM. A Comparative Review of Melanocytic Neoplasms. Veterinary Pathology 2002;39(6) 651-78. - 5.
Aronsohn MG, Carpenter JL. Distal extremity melanocytic nevi and malignant melanomas in dogs. Journal of American Animal Hospital Association 1990;26 605-12. - 6.
BoVeterinary Pathologylon B, Calderwood Mays MB, Hall BJ. Characteristics of canine melanomas and comparison of histology and DNA ploidy to their biologic effect. Veterinary Pathology 1990;27 96-102. - 7.
Smith SH, Goldschmidt MH, McManus PM. A comparative review of melanocytic neoplasms. Veterinary Pathology 2002;39(6) 651-78. - 8.
Spangler WL, Kass PH. The histologic and epidemiologic bases for prognostic considerations in canine melanocytic neoplasia. Veterinary Pathology 2006;43(2) 136-49. - 9.
Trappler MC, Popovitch CA, Goldschmidt MH, Goldschmidt KH, Risbon RE. Scrotal tumors in dogs: A retrospective study of 676 cases (1986–2010). Canadian Veterinary Journal 2014;55. - 10.
Ramos-Vara J, Beissenherz M, Miller M, Johnson G, Pace L, Fard A, Kottler S. Retrospective study of 338 canine oral melanomas with clinical, histologic, and immunohistochemical review of 129 cases. Veterinary Pathology 2000;37(6) 597-608. - 11.
Kim D, Royal A, Villamil J. Disseminated melanoma in a dog with involvement of leptomeninges and bone marrow. Veterinary Pathology 2009;46(1) 80-3. - 12.
Lacroux C, Raymon-Letron I, Bourges-Abella N, Lucas MN, Deviers A, Serra F, Degorce-Rubiales F, Delverdier M. Study of canine cutaneous melanocytic tumours: evaluation of histological and immunohistochemical prognostic criteria in 65 cases. Revue Médicine Véterinaire 2012;163(8-9) 393-401. - 13.
Smedley RC, Spangler WL, Esplin DG, Kitchell BE, Bergman PJ, Ho HY, Bergin IL, Kiupel M. Prognostic markers for canine melanocytic neoplasms: a comparative review of the literature and goals for future investigation. Veterinary Pathology 2011;48(1) 54-72. - 14.
Morrison WB. Tumors of the Skin and Subcutis. In: Morrison WB. (ed.) Cancer in Dogs and Cats: Medical and Surgical Management. University of Minnesota: Williams & Wilkins; 1998. p 499-500. - 15.
Millanta F, Fratini F, Corazza M, Castagnaro M, Zappulli V, Poli A. Proliferation activity in oral and cutaneous canine melanocytic tumors: correlation with histological parameters, location, and clinical behaviour. Research in Veterinary Sciences 2002;73 45-51. - 16.
Sulaimon SS, Kitchell BE, Ehrhart EJ. Immunohistochemical Detection of Melanoma-specific Antigens in Spontaneous Canine Melanoma. Journal of Comparative Pathology 2002;127(2–3) 162-8. - 17.
Banks W, Morris E. Results of radiation treatment of naturally occurring animal tumors. Journal of the American Veterinary Medical Association 1975;166(11) 1063. - 18.
Frimberger AE, Moore AS, Cincotta L, Cotter SM, Foley JW. Photodynamic therapy of naturally occurring tumors in animals using a novel benzophenothiazine photosensitizer. Clinical Cancer Research 1998;4(9) 2207-18. - 19.
Gillette EL. Hyperthermia effects in animals with spontaneous tumors. National Cancer Institute monograph 1982;61 361-4. - 20.
Marmor JB, Pounds D, Hahn N, Hahn GM. Treating spontaneous tumors in dogs and cats by ultrasound-induced hyperthermia. International Journal of Radiation Oncology Biology Physics 1978;4(11–12) 967-73. - 21.
Westberg S, Sadeghi A, Svensson E, Segall T, Dimopoulou M, Korsgren O, Hemminki A, Loskog AS, Totterman TH, von Euler H. Treatment efficacy and immune stimulation by AdCD40L gene therapy of spontaneous canine malignant melanoma. J Immunother 2013;36(6) 350-8. - 22.
Dow SW, Elmslie RE, Willson AP, Roche L, Gorman C, Potter TA. In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma. Journal of Clinical Investigation 1998;101(11) 2406. - 23.
MacEwen EG, Kurzman ID, Vail DM, Dubielzig RR, Everlith K, Madewell BR, Rodriguez CO, Phillips B, Zwahlen CH, Obradovich J. Adjuvant therapy for melanoma in dogs: results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor. Clinical Cancer Research 1999;5(12) 4249-58. - 24.
Bergman PJ, McKnight J, Novosad A, Charney S, Farrelly J, Craft D, Wulderk M, Jeffers Y, Sadelain M, Hohenhaus AE, Segal N, Gregor P, Engelhorn M, Riviere I, Houghton AN, Wolchok JD. Long-Term Survival of Dogs with Advanced Malignant Melanoma after DNA Vaccination with Xenogeneic Human Tyrosinase: A Phase I Trial. Clinical Cancer Research 2003;9(4) 1284-90. - 25.
Vail DM, Withrow SJ. Chapter 18 - Tumors of the Skin and Subcutaneous Tissues. In: Withrow SJ, Vail DM. editors. Withrow & MacEwen's Small Animal Clinical Oncology (Fourth Edition). Saint Louis: W.B. Saunders; 2007. p 375-401. - 26.
Smedley R, Spangler W, Esplin D, Kitchell B, Bergman P, Ho H-Y, Bergin I, Kiupel M. Prognostic Markers for Canine Melanocytic Neoplasms A Comparative Review of the Literature and Goals for Future Investigation. Veterinary Pathology 2011;48(1) 54-72. - 27.
Schultheiss PC. Histologic Features and Clinical Outcomes of Melanomas of Lip, Haired Skin, and Nail Bed Locations of Dogs. Journal of Veterinary Diagnostic Investigation 2006;18(4) 422-5. - 28.
Orkin M, Schwartzman RM. A Comparative Study of Canine and Human Dermatology. The Journal of Investigative Dermatology 1959;32 451-66. - 29.
de Camargo LP, Conceição LG, dos Santos Costa PR. Neoplasias melanocíticas cutâneas em cães: estudo retrospectivo de 68 casos (1996-2004). Brazilian Journal of Veterinary Research and Animal Science 2008;45(2) 138-52. - 30.
Goldschmidt M, Hendrick M. Tumors of the skin and soft tissues. Tumors in Domestic Animals, Fourth Edition 2008 45-117. - 31.
Spangler W, Kass P. The histologic and epidemiologic bases for prognostic considerations in canine melanocytic neoplasia. Veterinary Pathology 2006;43(2) 136-49. - 32.
Junqueira L, Carneiro J. Pele e Anexos. Histologia Básica. Rio de Janeiro, RJ, Brazil 2004. p 359-70. - 33.
Marks R. Epidemiology of melanoma. Clinical and Experimental Dermatology 2000;25(6) 459-63. - 34.
Valentine B, McManus P, Knox A. Malignant transformation of a giant congenital pigmented nevus (hamartoma) in a dog. Veterinary Dermatology (United Kingdom) 1999. - 35.
Conroy JD. Melanocytic Tumors of Domestic Animals: With Special Reference to Dogs. Archives of Dermatology 1967;96(4) 372-80. - 36.
Mulligan R. Melanoblastic tumors in the dog. American Journal of Veterinary Research 1961;22 345-51. - 37.
Koenig A, Bianco S, Fosmire S, Wojcieszyn J, Modiano J. Expression and significance of p53, rb, p21/waf-1, p16/ink-4a, and PTEN tumor suppressors in canine melanoma. Veterinary Pathology 2002;39(4) 458-72. - 38.
Dobson JM. Breed-predispositions to cancer in pedigree dogs. International Scholarly Research Notices 2013;2013. - 39.
Hsu M-Y, Meier FE, Nesbit M, Hsu J-Y, Van Belle P, Elder DE, Herlyn M. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors. The American Journal of Pathology 2000;156(5) 1515-25. - 40.
Meier F, Satyamoorthy K, Nesbit M, Hsu M-Y, Schittek B, Garbe C, Herlyn M. Molecular events in melanoma development and progression. Front Biosci 1998;3(D) 1005-10. - 41.
Seiter S, Schadendorf D, Herrmann K, Schneider M, Rösel M, Arch R, Tilgen W, Zöller M. Expression of CD44 variant isoforms in malignant melanoma. Clinical Cancer Research 1996;2(3) 447-56. - 42.
Goldschmidt MH, Hendrick MJ. Tumors of the Skin and Soft Tissues. In: M. DJ. (ed.) Tumors in Domestic Animals. Fourth Edition ed. Iowa State: Iowa State Press; 2002. p 78-83. - 43.
Gross TL, Ihrke PJ, Walder EJ, Affolter VK. Melanocytic tumors. In: Gross TL, Ihrke PJ, Walder EJ, Affolter VK. editors. Skin Diseases of the Dog and Cat: Blackwell Science Ltd; 2005. p 813-36. - 44.
Campagne C, Jule S, Alleaume C, Bernex F, Ezagal J, Chateau-Joubert S, Estrada M, Aubin-Houzelstein G, Panthier JJ, Egidy G. Canine melanoma diagnosis: RACK1 as a potential biological marker. Veterinary Pathology 2013;50(6) 1083-90. - 45.
Ginn PE, Mensett JEKL, Rukich PM. The skin and appendages. In: Grant M. (ed.) Jubb, Kennedy & Palmer's Pathology of Domestic Animals. Ontario, Canada: Grant, M.; 2007. p 759-60. - 46.
Friedrichs KR, Young KM. Diagnostic cytopathology in clinical oncology. Withrow and MacEwen's Small Animal Clinical Oncology 2013 111. - 47.
Daskalopoulou D, Gourgiotou K, Thodou E, Vaida S, Markidou S. Rapid cytological diagnosis of primary skin tumours and tumour-like conditions. Acta Dermato-Venereologica 1997;77(4) 292-5. - 48.
Dey P, Mallik M, Gupta S, Vasishta R. Role of fine needle aspiration cytology in the diagnosis of soft tissue tumours and tumour‐like lesions. Cytopathology 2004;15(1) 32-7. - 49.
Simeonov RS. The accuracy of fine-needle aspiration cytology in the diagnosis of canine skin and subcutaneous masses. Comparative Clinical Pathology 2012;21(2) 143-7. - 50.
Ghisleni G, Roccabianca P, Ceruti R, Stefanello D, Bertazzolo W, Bonfanti U, Caniatti M. Correlation between fine‐needle aspiration cytology and histopathology in the evaluation of cutaneous and subcutaneous masses from dogs and cats. Veterinary Clinical Pathology 2006;35(1) 24-30. - 51.
Liu W, Bennet M, Helm T. Canine melanoma: a comparison with human pigmented epithelioid melanocytoma. International Journal of Dermatology 2011;50 1542-5. - 52.
Cangul IT, van Garderen E, van der Linde-Sipman JS, van den Ingh TS, Schalken JA. Canine balloon and signet-ring cell melanomas: a histological and immunohistochemical characterization. Journal of Comparative Pathology 2001;125(2-3) 166-73. - 53.
Goldschmidt MH, Dustan RW, Stannard AA, von Tscharner C, Walder EJ, Yager JA. Melanocytic tumors and tumor-like lesions. In: Goldschmidt MH, Dustan RW, Stannard AA, von Tscharner C, Walder EJ, Yager JA. editors. Washington DC: Armed Forces Institute of Pathology; 1998. p 38-48. - 54.
Millanta F, Fratini F, Corazza M, Castagnaro M, Zappulli V, Poli A. Proliferation activity in oral and cutaneous canine melanocytic tumours: correlation with histological parameters, location, and clinical behaviour. Research in Veterinary Sciences 2002;73(1) 45-51. - 55.
Laprie C, Abadie J, Amardeilh MF, Net JLLE, Lagadic M, Delverdier M. MIB-1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma. Veterinary Dermatology 2001;12(3) 139-47. - 56.
De los Monteros AE, de las Mulas JM, Fernández A, Orós J, Rodríguez F. Immunohistopathologic Characterization of a Dermal Melanocytoma-Acanthoma in a German Shepherd Dog. Veterinary Pathology 2000;37(3) 268-71. - 57.
USDA licenses DNA vaccine for treatment of melanoma in dogs. Journal of American Veterinary Medical Association 2010;236(5) 495. - 58.
Koenig A, Wojcieszyn J, Weeks BR, Modiano JF. Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma. Veterinary Pathology 2001;38(4) 427-35. - 59.
Sulaimon S, Kitchell B, Ehrhart E. Immunohistochemical detection of melanoma-specific antigens in spontaneous canine melanoma. Journal of Comparative Pathology 2002;127(2-3) 162-8. - 60.
Ramos-Vara JA, Beissenherz ME, Miller MA, Johnson GC, Pace LW, Fard A, Kottler SJ. Retrospective study of 338 canine oral melanomas with clinical, histologic, and immunohistochemical review of 129 cases. Veterinary Pathology 2000;37(6) 597-608. - 61.
Sandusky GE, Jr., Carlton WW, Wightman KA. Immunohistochemical staining for S100 protein in the diagnosis of canine amelanotic melanoma. Veterinary Pathology 1985;22(6) 577-81. - 62.
Smedley RC, Lamoureux J, Sledge DG, Kiupel M. Immunohistochemical diagnosis of canine oral amelanotic melanocytic neoplasms. Veterinary Pathology 2011;48(1) 32-40. - 63.
Rieger E, Hofmann-Wellenhof R, Soyer HP, Kofler R, Cerroni L, Smolle J, Kerl H. Comparison of proliferative activity as assessed by proliferating cell nuclear antigen (PCNA) and Ki-67 monoclonal antibodies in melanocytic skin lesions. A quantitative immunohistochemical study. Journal of Cutaneous Pathology 1993;20(3) 229-36. - 64.
Millanta F, Fratini F, Corazza M, Castagnaro M, Zappulli V, Poli A. Proliferation activity in oral and cutaneous canine melanocytic tumours: correlation with histological parameters, location, and clinical behaviour. Research in Veterinary Sciences 2002;73(1) 45-51. - 65.
Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. International Journal of Cancer 1983;31(1) 13-20. - 66.
Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. American Journal of Pathology 1991;138(4) 867-73. - 67.
Laprie C, Abadie J, Amardeilh MF, Net JL, Lagadic M, Delverdier M. MIB-1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma. Veterinary Dermatology 2001;12(3) 139-47. - 68.
Roels S, Tilmant K, Ducatelle R. PCNA and Ki67 proliferation markers as criteria for prediction of clinical behaviour of melanocytic tumours in cats and dogs. Journal of Comparative Pathology 1999;121(1) 13-24. - 69.
Grollino MG, Cavallo D, Di Silverio F, Rocchi M, De Vita R. Interphase cytogenetics and flow cytometry analyses of renal tumours. Anticancer Research 1993;13(6A) 2239-44. - 70.
Yamal JM, Cox D, Hittelman WN, Boiko I, Malpica A, Guillaud M, MacAulay C, Follen M, Vlastos AT. Quantitative histopathology and chromosome 9 polysomy in a clinical trial of 4-HPR. Gynecology Oncology 2004;94(2) 296-306. - 71.
Kronenwett U, Castro J, Roblick UJ, Fujioka K, Ostring C, Faridmoghaddam F, Laytragoon-Lewin N, Tribukait B, Auer G. Expression of cyclins A, E and topoisomerase II alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination. BMC Cell Biology 2003;4 8. - 72.
Sidoni A, Cavaliere A, D'Amico GA, Brachelente G, Bucciarelli E. Biopathological significance of single cell DNA aneuploidy measured by static cytometry in breast cancer. Breast 2001;10(4) 325-9. - 73.
Li R, Sonik A, Stindl R, Rasnick D, Duesberg P. Aneuploidy vs. gene mutation hypothesis of cancer: recent study claims mutation but is found to support aneuploidy. Proceedings of the National Academy of Sciences USA 2000;97(7) 3236-41. - 74.
Postier RG, Lerner MR, Lightfoot SA, Vannarath R, Lane MM, Hanas JS, Brackett DJ. DNA ploidy and markovian analysis of neoplastic progression in experimental pancreatic cancer. Journal of Histochemistry Cytochemistry 2003;51(3) 303-9. - 75.
Elkowitz D, Daum F, Markowitz J, Proccaccino J, Boas E, Cuomo J, Kahn E. Risk factors for carcinoma of the pelvic ileal pouch/anal canal in ulcerative colitis. Annals of Clinical & Laboratory Science 2004;34(2) 143-9. - 76.
Cohen C. Image cytometric analysis in pathology. Human Pathology 1996;27(5) 482-93. - 77.
Nordemar S, Hogmo A, Lindholm J, Tani E, Sjostrom B, Auer G, Munck-Wikland E. The clinical value of image cytometry DNA analysis in distinguishing branchial cleft cysts from cystic metastases of head and neck cancer. Laryngoscope 2002;112(11) 1983-7. - 78.
Harada K, Nishizaki T, Ozaki S, Kubota H, Okamura T, Ito H, Sasaki K. Cytogenetic alterations in pituitary adenomas detected by comparative genomic hybridization. Cancer Genetics and Cytogenetics 1999;112(1) 38-41. - 79.
Frazier KS, Hines ME, 2nd, Hurvitz AI, Robinson PG, Herron AJ. Analysis of DNA aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry. Veterinary Pathology 1993;30(6) 505-11. - 80.
Lewis JE, Olsen KD, Sebo TJ. Carcinoma ex pleomorphic adenoma: pathologic analysis of 73 cases. Human Pathology 2001;32(6) 596-604. - 81.
Johnson TS, Raju MR, Giltinan RK, Gillette EL. Ploidy and DNA distribution analysis of spontaneous dog tumors by flow cytometry. Cancer Research 1981;41(8) 3005-9. - 82.
Bolon B, Calderwood Mays MB, Hall BJ. Characteristics of canine melanomas and comparison of histology and DNA ploidy to their biologic behavior. Veterinary Pathology 1990;27(2) 96-102. - 83.
Roels SL, Van Daele AJ, Van Marck EA, Ducatelle RV. DNA ploidy and nuclear morphometric variables for the evaluation of melanocytic tumors in dogs and cats. American Journal of Veterinary Research 2000;61(9) 1074-9. - 84.
Pilloni L, Bianco P, Difelice E, Cabras S, Castellanos ME, Atzori L, Ferreli C, Mulas P, Nemolato S, Faa G. The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions. European Journal of Histochemistry 2011;55(2) e20. - 85.
Yarden Y, Kuang WJ, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E, Schlessinger J, Francke U, Ullrich A. Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO Journal 1987;6(11) 3341-51. - 86.
Gomes J, Queiroga FL, Prada J, Pires I. Study of c-kit immunoexpression in canine cutaneous melanocytic tumors. Melanoma Research 2012;22(3) 195-201. - 87.
Foda HD, Zucker S. Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis. Drug Discovery Today 2001;6(9) 478-82. - 88.
Stamenkovic I. Extracellular matrix remodelling: the role of matrix metalloproteinases. Journal of Pathology 2003;200(4) 448-64. - 89.
Szabo KA, Ablin RJ, Singh G. Matrix metalloproteinases and the immune response. Clinical and Applied Immunology Reviews 2004;4(5) 295-319. - 90.
López-Otín C, Palavalli LH, Samuels Y. Protective roles of matrix metalloproteinases: From mouse models to human cancer. Cell Cycle 2009;8(22) 3657-62. - 91.
Cauwe B, Van den Steen PE, Opdenakker G. The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases. Critical Reviews in Biochemistry and Molecular Biology 2007;42 113-85. - 92.
Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Cell and Developmental Biology 2001;17 463-516. - 93.
Bergman RL. Matrix Metalloproteinases 2 and 9 in Normal Canine Cerebrospinal Fluid.Master thesis. Faculty of the Virginia Polytechnic Institute and State University 2001. - 94.
Björklund M, Koivunen E. Gelatinase-mediated migration and invasion of cancer cells. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 2005;1755(1) 37-69. - 95.
Visse R, Nagase H. Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases. Circulation Research 2003;92(8) 827-39. - 96.
Tonti Ga, Mannello F, Cacci E, Biagioni S. Neural stem cells at the crossroads: MMPs may tell the way. The International Journal of Developmental Biology 2009;53(1) 1-17. - 97.
Handsley MM, Edwards DR. Metalloproteinases and their inhibitors in tumor angiogenesis. International Journal of Cancer 2005;115(6) 849-60. - 98.
Galliera E, Tacchini L, Romanelli MM. Matrix metalloproteinases as biomarkers of disease: updates and new insights. Clin Chem Lab Med 2014. - 99.
Chaudhary A, Singh M, Bharti A, Asotra K, Sundaram S, Mehrotra R. Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck. Journal of Biomedical Science 2010;17(10). - 100.
Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nature Reviews Cancer 2009;9(4) 239-52. - 101.
Mannello F, Medda V. Nuclear localization of Matrix metalloproteinases. Progress in Histochemistry and Cytochemistry 2012;47(1) 27-58. - 102.
Kessenbrock K, Plaks V, Werb Z. Matrix Metalloproteinases: Regulators of the Tumor Microenvironment. Cell 2010;141(1) 52-67. - 103.
Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Seminars in Cancer Biology 2000;10(6) 415-33. - 104.
Deryugina E, Quigley J. Matrix metalloproteinases and tumor metastasis. Cancer and Metastasis Reviews 2006;25(1) 9-34. - 105.
Pires I, Gomes J, Prada J, Pereira D, Queiroga FL. MMP-2 and MMP-9 Expression in Canine Cutaneous Melanocytic Tumours: Evidence of a Relationship with Tumoural Malignancy2013. In: Lester D. (ed.) Recent Advances in the Biology, Therapy and Management of Melanoma. Rijeka: InTech; 2013. - 106.
Docampo MJ, Cabrera J, Rabanal RM, Bassols A. Expression of matrix metalloproteinase-2 and -9 and membrane-type 1 matrix metalloproteinase in melanocytic tumors of dogs and canine melanoma cell lines. Am J Vet Res 2011;72(8) 1087-96. - 107.
Le Bitoux MA, Stamenkovic I. Tumor-host interactions: the role of inflammation. Histochemical Cell Biology 2008;130(6) 1079-90. - 108.
Guo C, Buranych A, Sarkar D, Fisher PB, Wang XY. The role of tumor-associated macrophages in tumor vascularization. Vascular Cell 2014;6(1) 2. - 109.
Owen JL, Mohamadzadeh M. Macrophages and chemokines as mediators of angiogenesis. Frontiers Physiology 2013;4 159. - 110.
Vinogradov S, Warren G, Wei X. Macrophages associated with tumors as potential targets and therapeutic intermediates. Nanomedicine (Lond) 2014;9(5) 695-707. - 111.
Chanmee T, Ontong P, Konno K, Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel) 2014;6(3) 1670-90. - 112.
Brocker EB, Zwadlo G, Suter L, Brune M, Sorg C. Infiltration of primary and metastatic melanomas with macrophages of the 25F9-positive phenotype. Cancer Immunology & Immunotherapy 1987;25(2) 81-6. - 113.
Bianchini F, Massi D, Marconi C, Franchi A, Baroni G, Santucci M, Mannini A, Mugnai G, Calorini L. Expression of cyclo-oxygenase-2 in macrophages associated with cutaneous melanoma at different stages of progression. Prostaglandins & Other Lipid Mediators 2007;83(4) 320-8. - 114.
Pires I, Prada J, Coelho L, Garcia A, Queiroga FL. Tumour-Associated Macrophages (TAMs) and Cox-2 Expression in Canine Melanocytic Lesions. In: Murph M. (ed.) Melanoma in the Clinic - Diagnosis, Management and Complications of Malignancy. Rijeka: InTech; 2011. - 115.
Carvalho MI, Pires I, Prada J, Queiroga FL. T-lymphocytic infiltrate in canine mammary tumours: clinic and prognostic implications. In Vivo 2011;25(6) 963-9. - 116.
Kim JH, Yu CH, Yhee JY, Im KS, Sur JH. Lymphocyte infiltration, expression of interleukin (IL) -1, IL-6 and expression of mutated breast cancer susceptibility gene-1 correlate with malignancy of canine mammary tumours. J Comp Pathol 2010;142(2-3) 177-86. - 117.
Monteiro P. Factores de prognóstico em melanomas em caninos. UTAD Vila Real; 2013. - 118.
Makrilia N, Kollias A, Manolopoulos L, Syrigos K. Cell adhesion molecules: role and clinical significance in cancer. Cancer Investigation 2009;27(10) 1023-37. - 119.
Patel SD, Chen CP, Bahna F, Honig B, Shapiro L. Cadherin-mediated cell-cell adhesion: sticking together as a family. Current Opinion Structural Biology 2003;13(6) 690-8. - 120.
Santos M. Estudo da Immunoexpressão da caderina E em Tumores Melanocíticos do Cão. UTAD Vila Real; 2012. - 121.
Han JI, Kim Y, Kim DY, Na KJ. Alteration in E-cadherin/beta-catenin expression in canine melanotic tumors. Veterinary Pathology 2013;50(2) 274-80. - 122.
O'Banion MK, Winn VD, Young DA. cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase. Proceedings of the National Academy of Sciences USA 1992;89(11) 4888-92. - 123.
Williams CS, DuBois RN. Prostaglandin endoperoxide synthase: why two isoforms? Am J Physiol 1996;270(3 Pt 1) G393-400. - 124.
Hla T, Bishop-Bailey D, Liu CH, Schaefers HJ, Trifan OC. Cyclooxygenase-1 and -2 isoenzymes. International Journal Biochemical Cell Biology 1999;31(5) 551-7. - 125.
Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, Lipsky PE. Cyclooxygenase in biology and disease. FASEB Journal 1998;12(12) 1063-73. - 126.
Paglia D, Dubielzig RR, Kado-Fong HK, Maggs DJ. Expression of cyclooxygenase-2 in canine uveal melanocytic neoplasms. American Journal of Veterinary Research 2009;70(10) 1284-90. - 127.
Pires I, Garcia A, Prada J, Queiroga FL. COX-1 and COX-2 expression in canine cutaneous, oral and ocular melanocytic tumours. Journal of Comparative Patholology 2010;143(2-3) 142-9. - 128.
Mohammed SI, Khan KN, Sellers RS, Hayek MG, DeNicola DB, Wu L, Bonney PL, Knapp DW. Expression of cyclooxygenase-1 and 2 in naturally-occurring canine cancer. Prostaglandins Leukocyt Essential Fatty Acids 2004;70(5) 479-83. - 129.
Martinez CM, Penafiel-Verdu C, Vilafranca M, Ramirez G, Mendez-Gallego M, Buendia AJ, Sanchez J. Cyclooxygenase-2 expression is related with localization, proliferation, and overall survival in canine melanocytic neoplasms. Veterinary Pathology 2011;48(6) 1204-11. - 130.
Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. Journal Biology Chemestry 1996;271(2) 603-6. - 131.
Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. Journal Cell Science 2001;114(Pt 5) 853-65. - 132.
Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell 1996;87(7) 1153-5. - 133.
Risau W. Mechanisms of angiogenesis. Nature 1997;386(6626) 671-4. - 134.
Ferrara N, Keyt B. Vascular endothelial growth factor: basic biology and clinical implications. EXS 1997;79 209-32. - 135.
Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocrine Reviews 1997;18(1) 4-25. - 136.
Marneros AG. Tumor angiogenesis in melanoma. Hematology Oncology Clinics of North America 2009;23(3) 431-46. - 137.
Einspahr JG, Thomas TL, Saboda K, Nickolof BJ, Warneke J, Curiel-Lewandrowski C, Ranger-Moore J, Duckett L, Bangert J, Fruehauf JP, Alberts DS. Expression of vascular endothelial growth factor in early cutaneous melanocytic lesion progression. Cancer 2007;110(11) 2519-27. - 138.
Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. American Journa Physiology Cell Physiology 2002;282(5) C947-70. - 139.
Mittal K, Ebos J, Rini B. Angiogenesis and the tumor microenvironment: vascular endothelial growth factor and beyond. Seminars Oncology 2014;41(2) 235-51. - 140.
Benazzi C, Al-Dissi A, Chau CH, Figg WD, Sarli G, de Oliveira JT, Gartner F. Angiogenesis in spontaneous tumors and implications for comparative tumor biology. Scientific World Journal 2014;2014 919570. - 141.
Ruhrberg C. Growing and shaping the vascular tree: multiple roles for VEGF. Bioessays 2003;25(11) 1052-60. - 142.
Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacological Reviews 2004;56(4) 549-80. - 143.
Goel HL, Mercurio AM. VEGF targets the tumour cell. Nature Reviews Cancer 2013;13(12) 871-82. - 144.
Baldwin ME, Stacker SA, Achen MG. Molecular control of lymphangiogenesis. Bioessays 2002;24(11) 1030-40. - 145.
Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ, Lawitts JA, Benjamin L, Tan X, Manseau EJ, Dvorak AM, Dvorak HF. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. Journal Experimental Medicine 2002;196(11) 1497-506. - 146.
Gabrilovich D, Ishida T, Oyama T, Ran S, Kravtsov V, Nadaf S, Carbone DP. Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood 1998;92(11) 4150-66. - 147.
Ohm JE, Gabrilovich DI, Sempowski GD, Kisseleva E, Parman KS, Nadaf S, Carbone DP. VEGF inhibits T-cell development and may contribute to tumor-induced immune suppression. Blood 2003;101(12) 4878-86. - 148.
Ohm JE, Carbone DP. VEGF as a mediator of tumor-associated immunodeficiency. Immunology Research 2001;23(2-3) 263-72. - 149.
Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii S. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. Journal Experimental Medice 2001;193(9) 1005-14. - 150.
Xie K, Wei D, Shi Q, Huang S. Constitutive and inducible expression and regulation of vascular endothelial growth factor. Cytokine Growth Factor Rev 2004;15(5) 297-324. - 151.
Taylor KH, Smith AN, Higginbotham M, Schwartz DD, Carpenter DM, Whitley EM. Expression of vascular endothelial growth factor in canine oral malignant melanoma. Veterinary Comparative Oncology 2007;5(4) 208-18. - 152.
Simonetti O, Lucarini G, Brancorsini D, Nita P, Bernardini ML, Biagini G, Offidani A. Immunohistochemical expression of vascular endothelial growth factor, matrix metalloproteinase 2, and matrix metalloproteinase 9 in cutaneous melanocytic lesions. Cancer 2002;95(9) 1963-70. - 153.
Bayer-Garner IB, Hough AJ, Jr., Smoller BR. Vascular endothelial growth factor expression in malignant melanoma: prognostic versus diagnostic usefulness. Modern Pathology 1999;12(8) 770-4. - 154.
Brychtova S, Bezdekova M, Brychta T, Tichy M. The role of vascular endothelial growth factors and their receptors in malignant melanomas. Neoplasma 2008;55(4) 273-9. - 155.
Vacca A, Ria R, Ribatti D, Bruno M, Dammacco F. Angiogenesis and tumor progression in melanoma. Recenti Prog Med 2000;91(11) 581-7. - 156.
Wang Y, Yao X, Ge J, Hu F, Zhao Y. Can Vascular Endothelial Growth Factor and Microvessel Density Be Used as Prognostic Biomarkers for Colorectal Cancer? A Systematic Review and Meta-Analysis. ScientificWorldJournal 2014;2014 102736. - 157.
Nico B, Benagiano V, Mangieri D, Maruotti N, Vacca A, Ribatti D. Evaluation of microvascular density in tumors: pro and contra. Histology Histopathology 2008;23(5) 601-7. - 158.
Fanelli M, Locopo N, Gattuso D, Gasparini G. Assessment of tumor vascularization: immunohistochemical and non-invasive methods. International Journal Biological Markers 1999;14(4) 218-31. - 159.
Cuitino MC, Massone AR, Idiart JR. Lack of prognostic significance of angiogenesis in canine melanocytic tumours. Journal of Comparative Pathology 2012;147(2-3) 147-52. - 160.
Mukaratirwa S, Chikafa L, Dliwayo R, Moyo N. Mast cells and angiogenesis in canine melanomas: malignancy and clinicopathological factors. Veterinary Dermatology 2006;17(2) 141-6.